The approach of combining cost-effective nanopore sequencing and emerging environmental DNA (eDNA) metabarcoding could prove to be a promising tool for biodiversity documentation, especially in Malaysia. Given the substantial funding constraints in recent years, especially in relation to the country's biodiversity, many researchers have been limited to conduct restricted research without extended monitoring periods, potentially hindering comprehensive surveys and could compromise the conservation efforts. Therefore, the present study aimed to evaluate the application of eDNA metabarcoding on freshwater fish using short reads generated through nanopore sequencing. This assessment focused on species detection in three selected rivers within the Endau Rompin Landscape in Malaysia. Additionally, the study compared levels of species detection between eDNA metabarcoding and conventional sampling methods, examined the effectiveness of primer choice, and applied both metabarcoding and shotgun sequencing to the eDNA approach. We successfully identified a total of 22 and 71 species with an identification threshold of >97% and >90%, respectively, through the MinION platform. The eDNA metabarcoding approach detected over 13% more freshwater fish species than when the conventional method was used. Notably, the distinction in freshwater fish detection between eDNA primers for 12S rRNA and cytochrome oxidase I was insignificant. The cost for eDNA metabarcoding proved to be more effective compared to conventional sampling with cost reduction at 33.4%. With favourable cost-effectiveness and increased species detection, eDNA metabarcoding could complement existing methods, enhance holistic diversity documentation for targeted habitats and facilitate effective conservation planning.
Mislabelling in fish products is a highly significant emerging issue in world fish trade in terms of health and economic concerns. DNA barcoding is an efficient sequencing-based tool for detecting fish species substitution but due to DNA degradation, it is in many cases difficult to amplify PCR products of the full-length barcode marker (~650 bp), especially in severely processed products. In the present study, a pair of universal primers targeting a 198 bp sequence of the mitochondrial 16s rRNA gene was designed for identification of fish species in the processed fish products commonly consumed in Malaysia. The specificity of the universal primers was tested by both in-silico studies using bioinformatics software and through cross-reaction assessment by practical PCR experiments against the DNA from 38 fish species and 22 other non-target species (animals and plants) and found to be specific for all the tested fish species. To eliminate the possibility of any false-negative detection, eukaryotic endogenous control was used during specificity evaluation. The developed primer set was validated with various heat-treated (boiled, autoclaved and microwaved) fish samples and was found to show high stability under all processing conditions. The newly developed marker successfully identified 92% of the tested commercial fish products with 96-100% sequence similarities. This study reveals a considerable degree of species mislabelling (20.8%); 5 out of 24 fish products were found to be mislabelled. The new marker developed in this work is a reliable tool to identify fish species even in highly processed products and might be useful in detecting fish species substitution thus protecting consumers' health and economic interests.
The present study was carried out to examine the species identification and phylogenetic relationships of groupers in Malaysia using mitochondrial Cytochrome c Oxidase I (COI) gene, commonly known as barcoding gene. A total of 63 individuals comprising 10 species from three genera were collected from the coastal areas of Johor, Kelantan, Pahang, Perak, Selangor and Terengganu. All the individuals were morphologically identified and molecular works involved polymerase chain reaction (PCR) and sequencing of COI barcoding fragment (655 base pairs). Results from the BLAST search showed that 55 sequences could be assigned to 10 grouper species with high percentage identity index (≥95% to 100%), while eight grouper individuals showed discrepancies in their taxonomic identification based on the morphology and the COI barcoding results. The histogram of distances showed that there was a clear-cut barcode gap present in the sequences indicating a clear separation between intraspecific and interspecific distances. The pairwise genetic distances showed lowest pairwise distance between P. leopardus and P. maculatus (4.4%), while the highest pairwise distance was between E. bleekeri and P. maculatus (23.5%), supporting their morphological and habitat similarities and differences. Phylogenetic analysis (Neighbor-Joining) showed the presence of two major clades (1) genus Epinephelus vs (2) genus Plectropomus and Cephalopholis). In conclusion, the present study has managed to show the accuracy of DNA barcoding method for species identification, and utilization of COI gene for phylogenetic study among groupers. ?
Epinephelus fuscoguttatus is a commercially important marine fish species in southeast Asia. Due to overfishing and water pollution, this species has been declared as near-threatened. Thus, to provide information to help maintain and preserve the species, microsatellites were developed, using an enriched genomic library method. Thirty individuals were collected from the hatchery of the Fishery Research Institute, Terengganu, Malaysia. These individuals, from four to six years old, originated from Sabah and are maintained in captive culture as broodstock. Genomic DNA was extracted from the fins of selected individuals that weighed 3-8 kg. Ten microsatellite loci were found to be polymorphic in this population, with 5 to 21 alleles per locus. Observed and expected heterozygosities ranged from 0.53 to 0.97 and 0.59 to 0.95, respectively. Only one locus deviated significantly from Hardy-Weinberg equilibrium and no significant linkage disequilibrium was found among the pairs of loci. These polymorphic microsatellite loci will be used by the Malaysian Fishery Research Institute for investigating genetic diversity and for developing breeding strategies.
Conservation is imperative for the Asian snakeheads Channa striata, as the species has been overfished due to its high market demand. Using maternal markers (mitochondrial cytochrome c oxidase subunit 1 gene (COI)), we discovered that evolutionary forces that drove population divergence did not show any match between the genetic and morphological divergence pattern. However, there is evidence of incomplete divergence patterns between the Borneo population and the populations from Peninsular Malaysia. This supports the claim of historical coalescence of C. striata during Pleistocene glaciations. Ecological heterogeneity caused high phenotypic variance and was not correlated with genetic variance among the populations. Spatial conservation assessments are required to manage different stock units. Results on DNA barcoding show no evidence of cryptic species in C. striata in Malaysia. The newly obtained sequences add to the database of freshwater fish DNA barcodes and in future will provide information relevant to identification of species.
Morphological identification of fish taxa can sometimes prove difficult because phenotypic variation is either being affected by environmental factors, phenotypic characters are highly conserved or marker selection has been inappropriate. DNA based markers especially neutral mitochondrial DNA (mtDNA) have been used widely in recent times to provide better resolution of systematic relationships among vertebrate taxa. The Asian Arowana (Scleropages formosus) is a high value ornamental fish belonging to the family Osteoglossidae with a number of different colour variants distributed geographically across different locations around Southeast Asia. Systematic relationships among colour variants still remain unresolved. Partial sequences of the Cytochrome B (Cyt B) and DNA barcoding gene, Cytochrome C Oxidase I (COI) were used here to assess genetic relationships among colour variants and as a tool for molecular identification for differentiating among colour variants in this species. Results of the study show that in general, colour pattern shows no relationship with extent of COI or Cyt B mtDNA differentiation and so cannot be used to identify taxa. Partial sequences of the mtDNA genes were sufficient however, to identify S. formosus from a closely related species within the order Osteoglossidae.
Malaysian arowana (dragonfish; Scleropages formosus) is an ancient osteoglossid fish from southeast Asia. Due to the high demand of the ornamental fish trade and because of habitat loss, the species is close to extinction. We isolated and characterized 10 polymorphic microsatellites of this species, using 5'-anchored PCR. The number of alleles at the 10 microsatellite loci ranged from 2 to 28, with a mean of 7.8/locus. The observed heterozygosity ranged from 0.03 to 0.93 (mean: 0.39), whereas the expected heterozygosity ranged from 0.03 to 0.94 (mean: 0.46). Seven microsatellites deviated from Hardy-Weinberg equilibrium, and three conformed to Hardy-Weinberg equilibrium and were in linkage equilibrium. These 10 novel microsatellites should facilitate studies of genetic diversity and population structure of arowana to help plan actions for the conservation of the indigenous Malaysian arowana.
Invasive alien fish species have become a silent treat towards the ecosystem especially the native fish population in Malaysia. There has been a need to develop rapid identification methods that can aid management teams in identifying fish species that are not native to our ecosystem. Current visual identification methods are highly tedious and require time, delaying action towards curbing the invasion. The LAMP assay successfully identified six popular invasive fish species in Malaysia. None of the LAMP assays showed false positives and the Limit of Detection of the LAMP primers were highly sensitive and could detect DNA samples up to 1 × 10-15 ng/μl. The LAMP primers designed were highly specific to the target species and did not amplify non target species. DNA sequencing was done to ensure the accuracy of LAMP assay results. This study demonstrates that LAMP is a suitable tool in species identification efforts of invasive fish species in Malaysia.
We examine genetic structuring in three commercially important species of the teleost family Carangidae from Malaysian waters: yellowtail scad Atule mate, bigeye scad Selar crumenophthalmus and yellowstripe scad Selaroides leptolepis, from the Indo-Malay Archipelago. In view of their distribution across contrasting habitats, we tested the hypothesis that pelagic species display less genetic divergence compared with demersal species, due to their potential to undertake long-distance migrations in oceanic waters. To evaluate population genetic structure, we sequenced two mitochondrial (mt)DNA [650 bp of cytochrome oxidase I (coI), 450 bp of control region (CR)] and one nuclear gene (910 bp of rag1) in each species. One hundred and eighty samples from four geographical regions within the Indo-Malay Archipelago including a population of yellowtail from Kuwait were examined. Findings revealed that the extent of genetic structuring among populations in the semi-pelagic and pelagic, yellowtail and bigeye were lower than demersal yellowstripe, consistent with the hypothesis that pelagic species display less genetic divergence compared with demersal species. The yellowtail phylogeny identified three distinct clades with bootstrap values of 86%-99% in mtDNA and 63%-67% in rag1. However, in bigeye, three clades were also observed from mtDNA data while only one clade was identified in rag1 dataset. In yellowstripe, the mtDNA tree was split into three closely related clades and two clades in rag1 tree with bootstraps value of 73%-99% and 56% respectively. However, no geographic structure appears in both mtDNA and rag1 datasets. Hierarchical molecular variance analysis (AMOVA), pair wise FST comparisons and the nearest-neighbour statistic (Snn ) showed significant genetic differences among Kuwait and Indo-Malay yellowtail. Within the Indo-Malay Archipelago itself, two distinct mitochondrial lineages were detected in yellowtail suggesting potential cryptic species. Findings suggests varying degrees of genetic structuring, key information relevant to management of exploited stocks, though more rapidly evolving genetic markers should be used in future to better delimit the nature and dynamics of putative stock boundaries.
Tenualosa ilisha was found recently in the Perak River in western Peninsular Malaysia. Molecular phylogenetic and haplotype network analyses suggest that T. ilisha has two genetically distinct populations/groups: (i) Peninsular Malaysia (Malaysia population), and (ii) Peninsular Malaysia, Thailand, India and Bangladesh (Indian Ocean population). The results also suggest that the T ilisha population in Peninsular Malaysia is genetically heterogeneous with a typical anadromous migration pattern.
For centuries, morphology-based fish identification has been applied without molecular evaluation. Many studies showed that specimens with a similar morphology are frequently found to be quite genetically distinct. One of the fish species that still remains taxonomically problematic is a commercial snapper species, Lutjanus johnii. Because of morphological ambiguities among local fish taxonomists in Malaysia, we examined the ability of the cytochrome oxidase I (COI) gene to genetically examine the taxonomic status of L. johnii. A 626-base pair COI region was successfully amplified and aligned with conspecific sequences that were retrieved from GenBank. The phylogenetic tree obtained showed two major clusters; the first cluster consists of L. johnii from Straits of Malacca, Thailand, Australia, and China while the second cluster comprises L. johnii from China and India. The latter group showed sequence divergence greater than 3.5%. After observing this, we suspected that there might be a cryptic species between the South China Sea and Indian Ocean. This is the first molecular report concerning the commercial species of snapper, L. johnii, in Malaysia, which had only gained provisional recognition from morphological examination.
The complete mitogenome of the ray Taeniura lymma was recovered from genome skimming using the HiSeq sequencing system. The T. lymma mitogenome has 17,652 base pairs (59.13% A + T content) made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs and a 1906 bp non-coding AT-rich region. This mitogenome sequence is the second for a ray from Australian waters, the first for the genus Taeniura and the ninth for the family Dasyatidae.
To satisfy increasing demands for fish as food, progress must occur towards greater aquaculture productivity whilst retaining the wild and farmed genetic resources that underpin global fish production. We review the main selection methods that have been developed for genetic improvement in aquaculture, and discuss their virtues and shortcomings. Examples of the application of mass, cohort, within family, and combined between-family and within-family selection are given. In addition, we review the manner in which fish genetic resources can be lost at the intra-specific, species and ecosystem levels and discuss options to best prevent this. We illustrate that fundamental principles of genetic management are common in the implementation of both selective breeding and conservation programmes, and should be emphasized in capacity development efforts. We highlight the value of applied genetics approaches for increasing aquaculture productivity and the conservation of fish genetic resources.
Rachycentron canadum (cobia) is a marine fish species of high economic value in aquaculture due to its fast growth rate and good feed conversion efficacy. Regrettably, the industry has been affected by significant setbacks from high mortality due to diseases. Consequently, an improved perception of innate immunity correlated to each mucosal-associated lymphoid tissue (MALT) in teleost fish is necessary to understand hosts' response towards infections better. The utilization of polysaccharides in seaweed to stimulate the immune system has gathered unprecedented attention. The present study examined the immunostimulatory effects of Sarcodia suae water extracts (SSWE) on in vivo gill-, gut- and skin-associated lymphoid tissues (GIALT, GALT, and SALT) via immersion and oral ingestions. The GIALT genes (TNF-α, Cox2, IL-1β, IL-6, IL-8, IL-17 A/F1-3, IL-11, IL-12, IL-15, IL-18, MHCIa, IgM, and IgT) except IL-10 recorded positive upregulations in a dose-dependent manner post 24 h immersion in SSWE, indicating the algae extract contained bioactive compounds that could stimulate the immune genes. The upregulation of IL-12, IL-15, and IL-18 in the gills and hindgut post-SSWE immersion indicated that the extract could promote Th1-related responses in the MALTs. The modulation of immune gene expressions in the feeding trial was less potent than in the SSWE immersion. These findings indicated that the SSWE stimulated robust immune responses in both the GIALT and GALT of cobia. This suggests that the SSWE could be further explored as an effective immersive stimulant for fish, enhancing their immune system against pathogens.
In this study, we first conducted a genome survey assay for Sillago sihama by Illumina sequencing platform, and then developed 15 polymorphic microsatellite loci in a wild population. A total of 129.46 Gb raw data were obtained, of which 115.07 Gb were clean data, with a sequencing depth of 179.3-folds. This genome was estimated to be 522.6 Mb in size, with the heterozygosity, repeat content and GC content being 0.63%, 21% and 44%. A total of 630,028 microsatellites were identified from the genome, of which, dinucleotide repeat was the most abundant (56.80%), followed by mononucleotide repeat (30.23%). Furthermore, 60 pairs of primers were designed and synthesized based on microsatellite sequences, of which 15 were polymorphic in a wild population. A total of 91 alleles were found, with an average of 6.07 per locus. Number of alleles, observed and expected heterozygosity per locus ranged from two to 13, from 0.250 to 0.862, and from 0.396 to 0.901, respectively. Twelve loci were highly informative (PIC > 0.5), and the others were medium informative (0.25
This study was established to test the hypothesis of whether the codon optimization of fish growth hormone gene (FGH) based on P. pastoris preferred codon will improve the quantity of secreted rFGH in culture supernatant that can directly be used as fish feed supplements. The optimized FGH coding sequence (oFGH) and native sequence (nFGH) of giant grouper fish (Epinephelus lanceolatus) were cloned into P. pastoris expression vector (pPICZαA) downstream of alcohol oxidase gene (AOX1) for efficient induction of extracellular rFGH by adding 1% of absolute methanol. The results showed that recombinant P. pastoris was able to produce 2.80 ± 0.27 mg of oFGH compared to 1.75 ± 0.25 of nFGH in one litre of culture supernatant. The total body weight of tiger grouper fingerlings fed with oFGH increased significantly at third (P < 0.05) and fourth weeks (P < 0.01) of four-week experiment period compared to those fed with nFGH. Both oFGH and nFGH significantly enhanced the final biomass and fish survival percentage. In conclusion, codon optimization of FGH fragment was useful to increase rFGH quantity in the culture supernatant of P. pastoris that can be directly used as fish feed supplements. Further studies are still required for large scale production of rFGH and practical application in aquaculture production.
Biodiversity surveys are crucial for monitoring the status of threatened aquatic ecosystems, such as tropical estuaries and mangroves. Conventional monitoring methods are intrusive, time-consuming, substantially expensive, and often provide only rough estimates in complex habitats. An advanced monitoring approach, environmental DNA (eDNA) metabarcoding, is promising, although only few applications in tropical mangrove estuaries have been reported. In this study, we explore the advantages and limitations of an eDNA metabarcoding survey on the fish community of the Merbok Estuary (Peninsular Malaysia). COI and 12S eDNA metabarcoding assays collectively detected 178 species from 127 genera, 68 families, and 25 orders. Using this approach, significantly more species have been detected in the Merbok Estuary over the past decade (2010-2019) than in conventional surveys, including several species of conservation importance. However, we highlight three limitations: (1) in the absence of a comprehensive reference database the identities of several species are unresolved; (2) some of the previously documented specimen-based diversity was not captured by the current method, perhaps as a consequence of PCR primer specificity, and (3) the detection of non-resident species-stenohaline freshwater taxa (e.g., cyprinids, channids, osphronemids) and marine coral reef taxa (e.g., holocentrids, some syngnathids and sharks), not known to frequent estuaries, leading to the supposition that their DNA have drifted into the estuary through water movements. The community analysis revealed that fish diversity along the Merbok Estuary is not homogenous, with the upstream more diverse than further downstream. This could be due to the different landscapes or degree of anthropogenic influences along the estuary. In summary, we demonstrated the practicality of eDNA metabarcoding in assessing fish community and structure within a complex and rich tropical environment within a short sampling period. However, some limitations need to be considered and addressed to fully exploit the efficacy of this approach.
The Merbok Estuary comprises one of the largest remaining mangrove forests in Peninsular Malaysia. Its value is significant as it provides important services to local and global communities. It also offers a unique opportunity to study the structure and functioning of mangrove ecosystems. However, its biodiversity is still partially inventoried, limiting its research value. A recent checklist based on morphological examination, reported 138 fish species residing, frequenting or subject to entering the Merbok Estuary. In this work, we reassessed the fish diversity of the Merbok Estuary by DNA barcoding 350 specimens assignable to 134 species initially identified based on morphology. Our results consistently revealed the presence of 139 Molecular Operational Taxonomic Units (MOTUs). 123 of them are congruent with morphology-based species delimitation (one species = one MOTU). In two cases, two morphological species share the same MOTU (two species = one MOTU), while we unveiled cryptic diversity (i.e. COI-based genetic variability > 2%) within seven other species (one species = two MOTUs), calling for further taxonomic investigations. This study provides a comprehensive core-list of fish taxa in Merbok Estuary, demonstrating the advantages of combining morphological and molecular evidence to describe diverse but still poorly studied tropical fish communities. It also delivers a large DNA reference collection for brackish fishes occurring in this region which will facilitate further biodiversity-oriented research studies and management activities.
The spotted sardinella, Amblygaster sirm (Walbaum, 1792), is a commercial sardine commonly caught in Malaysia. Lack of management of these marine species in Malaysian waters could lead to overfishing and potentially declining fish stock populations. Therefore, sustainable management of this species is of paramount importance to ensure its longevity. As such, molecular information is vital in determining the A. sirm population structure and management strategy. In the present study, mitochondrial DNA Cytochrome b was sequenced from 10 A. sirm populations: the Andaman Sea (AS) (two), South China Sea (SCS) (six), Sulu Sea (SS) (one), and Celebes Sea (CS) (one). Accordingly, the intra-population haplotype diversity (Hd) was high (0.91-1.00), and nucleotide diversity (π) was low (0.002-0.009), which suggests a population bottleneck followed by rapid population growth. Based on the phylogenetic trees, minimum spanning network (MSN), population pairwise comparison, and F ST,and supported by analysis of molecular variance (AMOVA) and spatial analysis of molecular variance (SAMOVA) tests, distinct genetic structures were observed (7.2% to 7.6% genetic divergence) between populations in the SCS and its neighboring waters, versus those in the AS. Furthermore, the results defined A. sirm stock boundaries and evolutionary between the west and east coast (which shares the same waters as western Borneo) of Peninsular Malaysia. In addition, genetic homogeneity was revealed throughout the SCS, SS, and CS based on the non-significant F STpairwise comparisons. Based on the molecular evidence, separate management strategies may be required for A. sirm of the AS and the SCS, including its neighboring waters.
The Japanese scad Decapterus maruadsi (Carangidae) is an economically important marine species in Asia but its exploitation shows signs of overfishing. To document its stock structure, a population genetic and phylogeographic study of several populations of this species from the central part of the Indo-West Pacific region was conducted using the mitochondrial cytochrome b gene. Genetic homogeneity within the Sundaland region's population, including Rosario (the Philippines) and Ranong (Andaman Sea) populations was revealed with low nucleotide diversity (π = 0.001-0.003) but high haplotype diversity (h = 0.503-0.822). In contrast, a clear genetic structure was observed between this group and the northern Vietnam populations as revealed by FST, AMOVA and SAMOVA, while the central Vietnam population of Khanh Hoa is an admixed group between the two differentiated regional populations. The neutrality and mismatch distribution analyses supported a demographic expansion of D. maruadsi in between last Pleistocene to early Holocene period which influenced present day distribution pattern. Contemporary factors such as oceanic currents and different life history traits are also believed to play significant roles in the observed population structure and biogeographical pattern. Based on these results, recommendations on how stocks of the Japanese scad should be managed are offered.